The morphogenetic significance of the first quartet micromeres for the development of the snailBithynia tentaculata

Summary The significance of the first quartet of micromeres for the morphogenesis ofBithynia — a polar lobe-forming gastropod-has been studied by deletion experiments. After removal of the whole first quartet at the 8-cell stage a dorsoventrally organized veliger larva is formed. Apparently, an interaction between the animal micromeres and a vegetal macromere, which is essential for the origin of a dorsoventral organization in equally cleaving gastropods, is not required in polar lobe forming eggs. It is concluded that in these eggs dorsoventrality is determined by segregation of the polar lobe. The embryos, in which the first quartet has been removed, never develop head structures. This indicates that the capacity to form head structures is restricted to the first quartet of micromeres. Deletion of a specific first quartet micromere (1a, 1b, 1c or 1d) showed, however, that the individual cells of this quartet are not strictly determined right from their origin. Frequently regulative development was observed after removal of individual first quartet cells.     

High-resolution fate map of the snail Crepidula fornicata: the origins of ciliary bands, nervous system, and muscular elements

The littorinimorph gastropod Crepidula fornicata shows a spiralian cleavage pattern and has been the subject of studies in experimental embryology, cell lineage, and the organization of the larval nervous system. To investigate the contribution of early blastomeres to the veliger larva, we used intracellular cell lineage tracers in combination with high-resolution confocal imaging. This study corroborates many features derived from other spiralian fate maps (such as the origins of the hindgut and mesoderm from the 4d mesentoblast), but also yields new findings, particularly with respect to the origins of internal structures, such as the nervous system and musculature that have never been described in detail. The ectomesoderm in C. fornicata is mainly formed by micromeres of the 3rd quartet (principally 3a and 3b), which presumably represents a plesiomorphic condition for molluscs. The larval central nervous system is mainly formed by the micromeres of the 1st and 2nd quartet, of which 1a, 1c, and 1d form the anterior apical ganglion and nerve tracks to the foot and velum, and 2b and 2d form the visceral loop and the mantle cell. Our study shows that both first and second velar ciliary bands are generated by the same cells that form the prototroch in other spiralians and apparently bear no homology to the metatroch found in annelids.     

Cell specification and the role of the polar lobe in the gastropod mollusc Crepidula fornicata

A small polar lobe forms at the first and second cleavage divisions in the gastropod mollusc Crepidula fornicata. These lobes normally fuse with the blastomeres that give rise to the D quadrant at the two- and four-cell stages (cells ultimately generating the 4d mesentoblast and D quadrant organizer). Significantly, removal of the small polar lobe had no noticeable effect on subsequent development of the veliger larva. The behavior of the polar lobe and characteristic early cell shape changes involving protrusion of the 3D macromere at the 24-cell suggest that the D quadrant is specified prior to the sixth cleavage division. On the other hand, blastomere deletion experiments indicate that the D quadrant is not determined until the time of formation of the 4d blastomere (mesentoblast). In fact, embryos can undergo regulation to form normal-appearing larvae if the prospective D blastomere or 3D macromere is removed. Removal of the 4d mesentoblast leads to highly disorganized, radial development. Removal of the first quartet micromeres at the 8-cell stage also leads to the development of radialized larvae. These findings indicate that the embryos of C. fornicata follow the mode of development exhibited by equal-cleaving spiralians, which involves conditional specification of the D quadrant organizer via inductive interactions, presumably from the first quartet micromeres.     

Fate maps of the first quartet micromeres in the gastropod Ilyanassa obsoleta.

Cell fate specification in the gastropod mollusc Ilyanassa obsoleta involves both cell autonomous and inductive mechanisms, which depend on determinants localized first in the polar lobe and then in the D quadrant of the embryo. A complete cell lineage is lacking for this embryo and is essential for a critical interpretation of previous experimental results and an analysis of the mechanisms at the molecular level. Lineages of the first quartet micromeres were followed using Lucifer Yellow dextran as a tracer. The tracer was injected into individual first quartet micromeres using iontophoresis and patterns of fluorescence were analyzed in the larva after 8 days of development. Fluorescence was limited to head structures, including eyes, tentacles and velum. Structures on the left side were derived from 1a and 1d micromeres; 1a gave rise to the left eye, including the lens. Right side structures were derived from the 1c micromere and 1b contributed to the apical plate between the eyes and symmetrically to both sides of the velum. First quartet lineage data are compared with results from previous cell ablation experiments and with lineage data from other species.     

THE EARLY DEVELOPMENT OF THE MARINE PROSOBRANCH NASSARIUS (HINIA) RETICULATUS (LINNAEUS)

The early development up to the end of gastrulation of the marineprosobranch Nassarius (Hinia) reticula-tus (Linnaeus) has beenexamined by scanning and transmission electron microscopy. Thesynchrony of the cleavage divisions ends with the formationof the second quartet. The somatoblast 2d and the mesen-toblast4d appear earlier than the other micromeres of their quartets.From the 16- to the 32-cell stage the turret cells have an outgrowth.At the end of cleavage, Nassarius shows a typical sterroblastula.During gastrulation, the epibolic encirclement of the yolk macromeresby the micromeres is produced by an active flattening of themicromere cap and at the end of gastrulation the blastoporeis completely closed. The nuclei of the four yolk macromeresmigrate from the animal cell regions towards the vegetativepole. The development of Nassarius reticulatus is very similarto that of the related species llyanassa obsoleta (Say). (Received 19 December 1990; accepted 18 August 1991)     

Embryonic development of the sea butterfly Desmopterus papilio (Gastropoda: Thecosomata)

The embryonic development of the thecosome Desmopterus papilio is described for the first time. The mature individual produced a round-shaped egg mass containing ca. 200 fertilised eggs. First cleavage was observed 15 min after the release of the egg mass. Embryos showed typical molluscan spiral cleavage: macromeres produced the first and second quartets of micromeres in clockwise and counterclockwise directions, respectively. A trochophore larva hatched from the egg capsule 28 h after the release of the egg mass. Thereafter, the larva secreted a primary shell at the posterior part, developing into the veliger stage. These findings may be useful for future work on postembryonic development, especially on the loss of the veliger shell, in the genus Desmopterus which is the only group of thecosome species without a shell in the adult stage.     

Conserved mechanism of dorsoventral axis determination in equal-cleaving spiralians

Many members of the spiralian phyla (i.e., annelids, echiurans, vestimentiferans, molluscs, sipunculids, nemerteans, polyclad turbellarians, gnathostomulids, mesozoans) exhibit early, equal cleavage divisions. In the case of the equal-cleaving molluscs, animal-vegetal inductive interactions between the derivatives of the first quartet micromeres and the vegetal macromeres specify which macromere becomes the 3D cell during the interval between fifth and sixth cleavage. The 3D macromere serves as a dorsal organizer and gives rise to the 4d mesentoblast. Even though it has been argued that this situation represents the ancestral condition among the Spiralia, these inductive events have only been documented in equal-cleaving molluscs. Embryos of the nemertean Cerebratulus lacteus also undergo equal, spiral cleavage, and the fate map of these embryos is similar to that of other spiralians. The role of animal first quartet micromeres in the establishment of the dorsal (D) cell quadrant was examined in C. lacteus by removing specific combinations of micromeres at the eight-cell stage. To follow the development of various cell quadrants, one quadrant was labeled with DiI at the four-cell stage, and specific first quartet micromeres were removed from discrete positions relative to the location of the labeled quadrant. The results indicate that the first quartet is required for normal development, as removal of all four micromeres prevented dorsoventral axis formation. In most cases, when either one or two adjacent first quartet micromeres were removed from one side of the embryo, the cell quadrant on the opposite side, with its macromere centered under the greatest number of the remaining animal micromeres, ultimately became the D quadrant. Twins containing duplicated dorsoventral axes were generated by removal of two opposing first quartet micromeres. Thus, any cell quadrant can become the D quadrant, and the dorsoventral axis is established after the eight-cell stage. While it is not yet clear exactly when key inductive interactions take place that establish the D quadrant in C. lacteus, contacts between the progeny of animal micromeres and vegetal macromeres are established during the interval between the fifth and sixth round of cleavage divisions (i.e., 32- to 64-cell stages). These findings argue that this mechanism of cell and axis determination has been conserved among equal-cleaving spiralians.     

Cleavage in<Emphasis Type="Italic"> Nemertoderma westbladi</Emphasis> (Nemertodermatida) and its phylogenetic significance

Recent phylogenetic analyses of ribosomal and protein coding nuclear genes place the marine worms within the Nemertodermatida as one of the oldest lineages among the bilaterian animals. We studied the early embryonic cleavage in Nemertoderma westbladi to provide the first account of nemertodermatid early development. Live embryos were studied with interference microscopy and fixed embryos were either sectioned or studied with confocal laser scanning microscopy. Initially the divisions in the embryo are radial, but then micromeres are shifted clockwise generating a spiral pattern. The four-cell stage is characterized by duets of macromeres and micromeres and thus resembles the duet cleavage reported from members of the Acoela. However, subsequent stages differ from the acoel duet pattern and also from quartet spiral cleavage. The optimization of the cleavage pattern on current phylogenetic hypotheses with Nemertodermatida and Acoela as early bilaterian branches is discussed.     

Analysis of ciliary band formation in the mollusc Ilyanassa obsoleta

Two primary ciliary bands, the prototroch and metatroch, are required for locomotion and in the feeding larvae of many spiralians. The metatroch has been reported to have different cellular origins in the molluscs Crepidula fornicata and Ilyanassa obsoleta, as well as in the annelid Polygordius lacteus, consistent with multiple independent origins of the spiralian metatroch. Here, we describe in further detail the cell lineage of the ciliary bands in the gastropod mollusc I. obsoleta using intracellular lineage tracing and the expression of an acetylated tubulin antigen that serves as a marker for ciliated cells. We find that the I. obsoleta metatroch is formed primarily by third quartet derivatives as well as a small number of second quartet derivatives. These results differ from the described metatrochal lineage in the mollusc C. fornicata that derives solely from the second quartet or the metatrochal lineage in the annelid P. lacteus that derives solely from the third quartet. The present study adds to a growing body of literature concerning the evolution of the metatroch and the plasticity of cell fates in homologous micromeres in spiralian embryos.     

Larval development in Cnidaria: A connection to bilateria?

Among the basal animal phyla, the Cnidaria display many characteristics similar to the Bilateria (the higher Metazoa). However, the relation of that outgroup phyla to the Bilateria is still equivocal. Additionally to morphological and genetic data, studies on cnidarian embryogenesis are essential to clarify the Cnidaria-Bilateria relationship. We analyzed cellular differentiation during planula larvae development of the jellyfish Podocoryne carnea. Within 24 to 30 h postfertilization, the diploblastic body structure and all cell types found in polyps have already differentiated in the larva. Whereas the differentiating smooth muscles, RFamide-positive nerve cells, or nematocytes (stinging cells) express no axial polarity, a newly discovered tyrosine-tubulin-positive nervous system develops gradually in repetitive patterns from anterior to posterior. These data demonstrate that part of the cnidarian nervous system develops from anterior to posterior in serially repeated patterns. This developmental mechanism seems to follow the bilaterian pattern and would have antedated the Cambrian explosion.     

Gastrulation in Calcareous Sponges: In Search of Haeckel's Gastraea

Haeckel's studies of development in calcareous sponges (1872)led him to develop the "Gastraea Theory," which proposes thatthe ancestral mode of germ layer formation, or gastrulation,was by invagination to produce a functional gut. His observationsthat gastrulation in the Calcarea occurs by invagination ofa ciliated larva upon settlement and metamorphosis were supportedby remarkable photomicrographs of the stage by Hammer in 1908.Although no later work found the same stage, these conceptsare repeated in texts today. We have re-examined embryogenesisand metamorphosis in Sycon sp. cf. S. raphanus in order to understandwhen gastrulation occurs. Almost all larvae settle on theirciliated anterior pole and metamorphose into a bilayered juvenilewhose interior cells rapidly differentiate into choanocytesand other cells of the young sponge. After a four-year searchwe have found the transitory stage shown by Hammer in whichthe anterior cells invaginate into the posterior half of thelarva. The hole closes and it is not until some days later thatthe sponge forms an osculum at its apical pole. To understandwhether invagination comprises gastrulation and if the holecan be considered to be a blastopore we have carried out a reviewof the literature dealing with this brief moment in calcaroneansponge development. Despite the intrigue of this type of metamorphosis,we conclude that gastrulation occurs earlier, during formationof the two cellular regions of the larva, and that metamorphosisinvolves the reorganization of these already differentiatedregions. Considering the pivotal position occupied by the Calcareaas the possible sister-group to all other Metazoa, these resultscall for a reassessment of germ layer formation and of the relationshipsof the primary germ layers among basal metazoan phyla.     

The origin of animal phyla and the new phylum Procoelomata

BergstrÖm, Jan 1989 07 15: The origin of animal phyla and the new phylum Procoelomata. Lethaia , Vol. 22, pp. 259–269. Oslo. ISSN 0024–1164.
A model of metazoan evolution presented previously (BergstrÖm 1986 in Zoologica Scripta 15 ) explains deuterostomian characters as derived from protostomian ones through loosening of the constraints in the spiralian type of morphogenesis. This fits phylogenies derived from studies of molecular sequences. The model helps explain (1) the well-known mixture of proto- and deuterostomian features in several groups; (2) the difficulties in making a phylogeny based on comparative anatomy, and (3) the fossil explosion in the Cambrian. Since protostomian features such as a ciliated locomotory sole and a pelagic larva with ciliary bands are widely distributed in branches of the phylogenetic tree, they must have been present in the stem of the tree. Most probably the stem forms were pseudosegmented, which helps explain how segmentation, oligomery and non-segmentation could evolve repeatedly in derived groups. Origination of new phyla involved macroevolutionary changes primarily in the mode of feeding and locomotion. The stem phylum, from which most other phyla appear to have been derived directly, is here named the Procoelomata. Machaeridian-type animals are referred to it. The Ediacaran-type Precambrian fossils Cannot be placed in the metazoan evolutionary tree. * Biochemical evolution, Cambrian fossil explosion, Deuterostomia, eukaryote evolution, Machaeridia. macroevolution, Precambrian fossils. Procoelomata, Protostomia .     

Embryogenesis in the glass sponge Oopsacas minuta: Formation of syncytia by fusion of blastomeres

Sponges (Porifera) are unusual animals whose body plans makeinterpreting phylogenetic relationships within the group andwith other basal metazoan taxa a difficult task. Although molecularapproaches have offered new insights, some questions requirea morphological approach using detailed ultrastructural or lightmicroscopical studies of developing embryos and larvae. Glasssponges (Hexactinellida) have perhaps the most unusual bodyplan within the Metazoa because the majority of the tissue ofthe adult consists of a single giant multinucleated syncytiumthat forms the inner and outer layers of the sponge and is joinedby cytoplasmic bridges to uninucleate cellular regions. Herewe have used serial section transmission and high-resolutionscanning electron microscopy to examine when syncytia firstform in the cave-dwelling glass sponge Oopsacas minuta. We confirmthat in O. minuta blastomeres are separate until the 32-cellstage; cleavage is equal but asynchronous until a hollow blastulais formed. The sixth division yields a collection of variouslysized micromeres at the surface of the embryo and large yolk-and lipid-filled macromeres lining the blastocoel. Syncytiathen form by the fusion of micromeres to form cytoplasmic bridgeswith each other and the fusion of macromeres to form the futuremultinucleated trabecular tissue of the larva and adult sponge.The multinucleated trabecular tissue envelops and forms cytoplasmicbridges with all uninucleate cells, covering the developinglarva with a continuous syncytial epithelium. Differentiationof tissues occurs very early during embryogenesis with the separationof uninucleate and multinucleate lineages, but all cells andsyncytia are joined by cytoplasmic bridges such that there iscytoplasmic continuity throughout the entire larva. Althoughglass sponges begin life as a cellular embryo, the unusual mechanismof syncytia formation at such an early stage in developmentdistinguishes this group of animals from their closest multicellularrelatives, the Demospongiae. Most important, however, thesedata lend support to the hypothesis that the original metazoanswere cellular, not syncytial.     

The Position of Arthropods in the Animal Kingdom: A Search for a Reliable Outgroup for Internal Arthropod Phylogeny

Morphological evidence for the phylogeny of the animal kingdom has been discussed by numerous authors. DNA sequencing and phylogenetic methods for analyzing these data are alternative approaches to animal phylogeny, but the phenomenon of long branch attraction and poor taxonomic sampling have caused misinterpretations of metazoan relationships. Here we report a cladistic approach to metazoan evolution including 133 18S rDNA sequences of 31 animal phyla. Despite the difficulties associated with analyzing large data sets, our data suggest that the Bilateria and Protostomia are monophyletic. The internal phylogeny of the protostomes is divided into two main clades. One clade includes the classical protostome worms (annelids, sipunculans, echiurans, pogonophorans, and vestimentiferans), mollusks, nemerteans, “lophophorates,” platyhelminths, rotiferans, and acanthocephalans, although the internal resolution of the clade is very low. The second clade includes arthropods and other molting animals: tardigrades, onychophorans, nematodes, nematomorphans, kinorhynchs, and priapulans. The arthropods and related phyla lack a ciliated larvae, lack a multiciliate (locomotory) epithelium, and share many features, notably, a reduced coelomic cavity and the presence of a cuticle which molts. The use of these outgroups within the molting clade to root arthropod phylogenies is recommended instead of using annelids or other spiralians. The data are quite conclusive in those phyla with a good taxonomic sampling (i.e., platyhelminths and arthropods).     

The embryonic value of the micromeres in Ilyanassa obsoleta,as determined by deletion experiments. II. The second quartet cells

Summary

Each of the second quartet micromeres of Ilyanassa obsoleta was removed and the effects on larval development analyzed. Structures most often affected by removal of 2a were the left velar lobe, the left eye and the left statolith. Removal of 2b resulted in no consistent pattern of defects. Removal of 2c resulted in atypical shell development, absence of the heart, and eversión of the stomodeum; additional effects noted in some individuals involved the right velar lobe, the right statolith and perhaps the right eye. Anomalous birefringent bodies appeared frequently in the anterior region of the larva, on the right side after removal of 2c, and on the left side after removal of 2a. After removal of 2d the external shell was usually absent or rudimentary, the stomodeum was often everted, and other effects were noted in some individuals. On the basis of the deletion experiments, each second quartet micromere is judged to have a different embryonic value.     

Origin and evolution of animal life cycles

The ‘origin of larvae’ has been widely discussed over the years, almost invariably with the tacit understanding that larvae are secondary specializations of early stages in a holobenthic life cycle. Considerations of the origin and early radiation of the metazoan phyla have led to the conclusion that the ancestral animal (= metazoan) was a holopelagic organism, and that pelago-benthic life cycles evolved when adult stages of holopelagic ancestors became benthic, thereby changing their life style, including their feeding biology. The literature on the larval development and phylogeny of animal phyla is reviewed in an attempt to infer the ancestral life cycles of the major animal groups. The quite detailed understanding of larval evolution in some echinoderms indicates that ciliary filter-feeding was ancestral within the phylum, and that planktotrophy has been lost in many clades. Similarly, recent studies of the developmental biology of ascidians have demonstrated that a larval structure, such as the tail of the tadpole larva, can easily be lost, viz. through a change in only one gene. Conversely, the evolution of complex structures, such as the ciliary bands of trochophore larvae, must involve numerous genes and numerous adaptations. The following steps of early metazoan evolution have been inferred from the review. The holopelagic ancestor, blastaea, probably consisted mainly of choanocytes, which were the feeding organs of the organism. Sponges may have evolved when blastaea-like organisms settled and became reorganized with the choanocytes in collar chambers. The eumetazoan ancestor was probably the gastraea, as suggested previously by Haeckel. It was holopelagic and digestion of captured particles took place in the archenteron. Cnidarians and ctenophores are living representatives of this type of organization. The cnidarians have become pelago-benthic with the addition of a sessile, adult polyp stage; the pelagic gastraea-like planula larva is retained in almost all major groups, but only anthozoans have feeding larvae. Within the Bilateria, two major lines of evolution can be recognized: Protostomia and Deuterostomia. In protostomes, trochophores or similar types are found in most spiralian phyla; trochophore-like ciliary bands are found in some rotifers, whereas all other aschelminths lack ciliated larvae. It seems probable that the trochophore was the larval type of the ancestral, pelago-benthic spiralian and possible that it was ancestral in all protostomes. Most of the non-chordate deuterostome phyla have ciliary filter-feeding larvae of the dipleurula type, and this strongly indicates that the ancestral deuterostome had this type of larva.     

Evolution of sensory structures in basal metazoa

Cnidaria have traditionally been viewed as the most basal animalswith complex, organ-like multicellular structures dedicatedto sensory perception. However, sponges also have a surprisingrange of the genes required for sensory and neural functionsin Bilateria. Here, we: (1) discuss "sense organ" regulatorygenes, including; sine oculis, Brain 3, and eyes absent, thatare expressed in cnidarian sense organs; (2) assess the sensoryfeatures of the planula, polyp, and medusa life-history stagesof Cnidaria; and (3) discuss physiological and molecular datathat suggest sensory and "neural" processes in sponges. We thendevelop arguments explaining the shared aspects of developmentalregulation across sense organs and between sense organs andother structures. We focus on explanations involving divergentevolution from a common ancestral condition. In Bilateria, distinctsense-organ types share components of developmental-gene regulation.These regulators are also present in basal metazoans, suggestingevolution of multiple bilaterian organs from fewer antecedentsensory structures in a metazoan ancestor. More broadly, wehypothesize that developmental genetic similarities betweensense organs and appendages may reflect descent from closelyassociated structures, or a composite organ, in the common ancestorof Cnidaria and Bilateria, and we argue that such similaritiesbetween bilaterian sense organs and kidneys may derive froma multifunctional aggregations of choanocyte-like cells in ametazoan ancestor. We hope these speculative arguments presentedhere will stimulate further discussion of these and relatedquestions.     

Back in time: a new systematic proposal for the Bilateria

Conventional wisdom suggests that bilateral organisms arose from ancestors that were radially, rather than bilaterally, symmetrical and, therefore, had a single body axis and no mesoderm. The two main hypotheses on how this transformation took place consider either a simple organism akin to the planula larva of extant cnidarians or the acoel Platyhelminthes (planuloid-acoeloid theory), or a rather complex organism bearing several or most features of advanced coelomate bilaterians (archicoelomate theory). We report phylogenetic analyses of bilaterian metazoans using quantitative (ribosomal, nuclear and expressed sequence tag sequences) and qualitative (HOX cluster genes and microRNA sets) markers. The phylogenetic trees obtained corroborate the position of acoel and nemertodermatid flatworms as the earliest branching extant members of the Bilateria. Moreover, some acoelomate and pseudocoelomate clades appear as early branching lophotrochozoans and deuterostomes. These results strengthen the view that stem bilaterians were small, acoelomate/pseudocoelomate, benthic organisms derived from planuloid-like organisms. Because morphological and recent gene expression data suggest that cnidarians are actually bilateral, the origin of the last common bilaterian ancestor has to be put back in time earlier than the cnidarian-bilaterian split in the form of a planuloid animal. A new systematic scheme for the Bilateria that includes the Cnidaria is suggested and its main implications discussed.     

MAPK activation and the specification of the D quadrant in the gastropod mollusc, Crepidula fornicata

Embryos of the gastropod snail Crepidula fornicata exhibit a typical spiral cleavage pattern. Although a small polar lobe is formed at the first and second cleavage divisions, the embryo of C. fornicata exhibits a mode of development similar to that of equal-cleaving spiralians in which the D quadrant is conditionally specified by inductive interactions involving the derivatives of the first quartet micromeres. This study demonstrates that mitogen activated protein kinases, MAPK, are initially activated in the progeny of the first quartet micromeres, just prior to the birth of the third quartet (e.g., late during the 16-cell and subsequently during the 20-cell stages). Afterwards, MAPK is activated in 3D just prior to the 24-cell stage, transiently in 4d and finally in a subset of animal micromeres immediately following those stages. This pattern of MAPK activation differs from that reported for other spiralians. Using an inhibitor of MAPK kinase (MEK), we demonstrated that activated MAPK is required for the specification of the 3D macromere, during the late 16-cell through early 24-cell stages. This corresponds to the interval when the progeny of the first quartet micromeres specify the D quadrant macromere. Activated MAPK is not required in 3D later during the 24-cell stage or in the embryonic organizer, 4d, for its normal activity. Likewise, activated MAPK is not required in the animal micromeres during subsequent stages of development. Additional experiments suggest that the polar lobe, though not required for normal development, may play a role in restricting the activation of MAPK and biasing the specification of the 3D macromere.